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Alteration of the Substrate Specificity of Benzoylformate Decarboxylase from Pseudomonas putida by Directed Evolution
Author(s) -
Lingen Bettina,
KolterJung Doris,
Dünkelmann Pascal,
Feldmann Ralf,
Grötzinger Joachim,
Pohl Martina,
Müller Michael
Publication year - 2003
Publication title -
chembiochem
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.05
H-Index - 126
eISSN - 1439-7633
pISSN - 1439-4227
DOI - 10.1002/cbic.200200475
Subject(s) - benzaldehyde , chemistry , pseudomonas putida , substrate (aquarium) , acetaldehyde , stereochemistry , enantiopure drug , cofactor , enzyme , catalysis , directed evolution , organic chemistry , mutant , biochemistry , enantioselective synthesis , ethanol , oceanography , gene , geology
Alteration of the substrate specificity of thiamin diphosphate (ThDP)‐dependent benzoylformate decarboxylase (BFD) by error‐prone PCR is described. Two mutant enzymes, L476Q and M365L‐L461S, were identified that accept ortho ‐substituted benzaldehyde derivatives as donor substrates, which leads to the formation of 2‐hydroxy ketones. Both variants, L476Q and M365L‐L461S, selectively catalyze the formation of enantiopure ( S )‐2‐hydroxy‐1‐(2‐methylphenyl)propan‐1‐one with excellent yields, a reaction which is only poorly catalyzed by the wild‐type enzyme. Different ortho ‐substituted benzaldehyde derivatives, such as 2‐chloro‐, 2‐methoxy‐, or 2‐bromobenzaldehyde are accepted as donor substrates by both BFD variants as well and conversion with acetaldehyde resulted in the corresponding ( S )‐2‐hydroxy‐1‐phenylpropan‐1‐one derivatives. As deduced from modeling studies based on the 3D structure of wild‐type BFD, reduction of the side chain size at position L461 probably results in an enlarged substrate binding site and facilitates the initial binding of ortho ‐substituted benzaldehyde derivatives to the cofactor ThDP.